Hepatitis C Virus Nonstructural Protein 5A (NS5A) Is an RNA-binding Protein

Abstract

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-␣ response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nM range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.

Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis

“…HCV NS5A is a phosphoprotein with no known enzymatic activity that plays an essential, although poorly understood, role in the viral life cycle. NS5A is required for RNA replication, for infectious HCV assembly, and for interactions with a variety of cellular proteins (22)(23)(24); the NS5A protein is also the target of the most potent anti-HCV inhibitors discovered to date (25). The largest TDAV NS5A insertion resides in the equivalent of HCV NS5A domain I, a zinc-binding region with RNA-binding activity (26,27); the relevance of the insertions in the TDAV life cycle is not known.…”

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…The cytoplasmic part of NS5A is composed of a well structured domain 1 (D1) and two intrinsically disordered domains (D2 and D3) that exist as highly dynamic interconverting conformers. NS5A-D1, which is the target of daclatasvir (7,17), contains a zinc finger motif and possesses RNA binding properties (18). Crystallographic studies revealed that this domain could adopt at least three different homodimeric conformations (19 -21), underscoring the multifunctionality of the protein.…”

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

“…The basic groove in the inner side of the claw is positioned away from the membrane and could interact with RNA (38). It was found that bacterially expressed NS5A binds to the 3Ј-end of HCV positive and negative strand RNA with a preference for the poly(U/UC) tract in the 3Ј-NTR of positive strand RNA (39). Conceivably, NS5A dimers might oligomerize into large two-dimensional assemblies forming an extended groove.…”